Protein phosphorylation and dephosphorylation is a key regulating mechanism of biological processes and therefore a post-translational modification of profound biological importance. It is of critical importance in intracellular signal transduction processes where defects in the kinase-phosphatase switch have been implicated as an important mechanism in several disease processes, including cancer.
The main phosphorylated amino acid residues formed by post-translational modifications are the following

A need has emerged in the Life Sciences for means to selectively extract phosphorylated peptides or proteins. Protein purification is one example where removal of phosphorylated biproducts presents serious challenges. Mapping of the phosphoproteome (defined here as the complete cellular repertoire of phosphorylated proteins and peptides) is an important objective. Apart from a fundamental understanding of disease processes, the objectives can be to identify and characterise new drug targets, to evaluate the efficacy of new drugs or to identify biomarkers for disease leading to new diagnostic tools.
In this context it has proven particularly difficult to obtain a comprehensive picture of the phosphorylated protein landscape due to the low abundance or difficulties in enrichment of such proteins from biological extracts or digests. In particular, proteins and peptides phosphorylated at tyrosine residues constitute a challenging analytical problem. As tyrosine phosphorylation is a sub-stoichiometric modification often occurring in low-abundance proteins, the presently used separation and detection techniques based on antibodies or chelating chromatographic materials often exhibit insufficient selectivity and sensitivity to allow the modified proteins to be individually determined. Thus, there is a general need for techniques capable of separating or sensing common structures in proteins or peptides. Apart from the need for a generic fractionation tool capable of isolating all pTyr-containing peptides over non-phosphorylated peptides and peptides phosphorylated at Ser (serine) or Thr (threonine), other levels of selectivity are equally important (FIG. 1). In addition to the need for pSer (phosphorylated serine) and pThr (phosphorylated threonine) selectivities, where also dedicated fractionation tools are needed, receptors that can recognize the amino acid sequence around the phosphorylation site would find use in diagnostic applications once reliable biomarkers have been identified. Such receptors could be incorporated in sensors where the binding event would be translated into a measurable signal. For instance, binding can give rise to a change in colour or luminescence of a receptor which could be easily measured.
Alternatively, such receptors if prepared in soluble or nanoparticulate form could be used as therapeutic agents e.g. inhibiting dephosphorylation events or as imaging agents provided that the receptor contains a visible label.
Several attempts to complex phosphorylated peptides have been based on low molecular weight artificial receptors but they often exhibit a charge dependent sequence bias due to their charged nature and hence preference for complementary charged amino acids. Neural receptors containing no charge bias would be more interesting in this regard. In this area molecularly imprinted polymers could play an important role, complementing currently used immunological and chemical methods.
Molecular imprinting has resulted in a range of robust polymer-based receptors (known as MIPs), predominantly for small lipophilic target molecules. The technique entails copolymerisation of mono- and di-functional monomers in the presence of a template, which is thereafter removed to leave sites that can be reoccupied by the template or closely related compounds. Vis-á-vis biological receptors, MIPs are distinguished by their robustness and ease of synthesis, which has led to their use in a range of molecular recognition based applications targeting small molecules.
While MIPs have proven their value for the enrichment of low molecular weight analytes, their use in the enrichment of peptide or protein target molecules has met with limited success.1 However, this may change by the use of epitopes of the target protein in question as templates. Striking affinities and selectivities have been observed for peptides2 and proteins3 using polymers imprinted with shorter peptide sequences derived from the N- or C-termini of the target proteins or peptides.